Antisense cDNA probes for Northerns

Patrick J. Moran pmoran at U.Arizona.edu
Tue Feb 9 11:35:41 EST 1999


Dear Colleagues:

I am a postdoctoral researcher at the University of Arizona, interested in
characterizing responses of plants to feeding by aphid insects, using
Arabidopsis as a model.  I am at my wit's end to get Northerns using
Arabidopsis leaves and inflorescence bolts to work. Gels and nylon membrane
blots with 10-20 ug total RNA look good, but then I get no signal on
autoradiograms, or nothing but background. I have been trying both
formamide and non-formamide hybridization buffers. These attempts have
involved mostly random-prime 32-P labeled, double-stranded, boil-denatured
cDNA (purified before labeling with the Prep-A-Gene kit). Recently I have
been trying to generate antisense DIG-labeled RNA transcripts as probes
using T7 polymerase, but I have been having problems with degradation. I've
seen papers involving various plants where antisense PCR-labeled cDNA
probes were used for Northerns. Does anyone have a protocol for generating
these probes from standard plasmids or purified fragments (PCR conditions,
yield evaluation afterwards, concentration to use in hybridization)?

I would greatly appreciate any help. Any general tips from people who do
Northerns with Arabidopsis would be great, too! The problems I am having
are especially vexing because I have generated good Northerns with
hypocotyls of a few other plants (cucurbits).

Thank You,

Patrick J. Moran, Ph.D.




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